Fig 1.
CRISPR screening identifies ANXA1 as a entry factor for PAstV infection.
(A) Schematic of genome-wide CRISPR screen for host factors required for PAstV infection. Illustration from NIAID NIH BioArt Source (//bioart.niaid.nih.gov). PK15 cells were transduced with a CRISPR knockout library and subjected to four rounds of PAstV infection. Surviving cells from rounds 2 and 4 were harvested and analyzed by sequencing (n = 2 biological replicates). (B) PK15 and PK15-Cas9 were infected at MOI 0.01 and imaged at 96 h. (C, D), Scatterplots depicting sgRNA enrichment in rounds 2 (C) and 4 (D) of PAstV screening. (E) Percentage of PAstV-infected cells (MOI = 1, 24 hpi) in indicated knockdown lines, assessed by immunostaining with mouse anti-Capsid antibody. Data are normalized to non-targeting (NT) controls and presented as mean ± SD (n = 4 independent experiments). Data represent mean ± SD (n = 3). Statistical significance by unpaired two-tailed Student’s t-test. (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).
Fig 2.
ANXA1 supports PAstV infection.
(A) Representative histograms of ANXA1 surface expression measured by flow cytometry. (B) Western blot confirming ANXA1 knockout (KO) in PK15 cells transduced with sgRNAs (n = 2). (C) Viability of ANXA1 KO PK15 cells assessed by CCK8 assay. Data represent mean ± SD (n = 3). (D) Viral titers in WT and ANXA1 KO PK15 at 24 hpi after infection at MOI 0.01. (E) Viral RNA in WT and ANXA1 KO PK15 at the indicated times after infection at MOI 0.01. (F) Viral proteins in WT and ANXA1 KO PK15 at 24 hpi after infection at MOI 0.01, by Western blot. (G) Immunofluorescence detection of PAstV infection (MOI = 0.01) in WT and ANXA1 KO PK15 cells. Nuclei stained with DAPI; virus stained with mouse anti-PAstV Capsid antibody. Scale bar, 50 μm. (H) Overview of virus binding and internalization assay. Illustration from NIAID NIH BioArt Source (//bioart.niaid.nih.gov). (I) Impact of ANXA1 deletion on PAstV binding to PK15 cells. (J) Confocal imaging of virus particles (MOI = 10) binding to WT and ANXA1 KO PK15 cells. Scale bar, 5 μm. (K) Competitive blocking of PAstV binding with ANXA1 polyclonal antibody (pAb). PK15 cells were blocked with ANXA1 pAb (5–50 μg/ml) for 1 h and incubated with PAstV (MOI = 10) at 4°C for 1 h, binding quantified by RT-qPCR. (L) Confocal images showing PAstV binding (MOI = 10) after blocking with ANXA1 pAb (50 μg/ml) or mouse IgG (50 µg/ml). Scale bar, 5 μm. Data represent mean ± SD (n = 3). Statistical significance by unpaired two-tailed Student’s t-test and Two-way ANOVA. (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).
Fig 3.
ANXA1 facilitates early PAstV entry and rescue restores susceptibility.
(A) Western blot validating ANXA1 overexpression in PK15 and IPEC-J2 cells using mouse anti-ANXA1 antibody. (B) RT-qPCR analysis of PAstV infection (MOI = 0.01) at 24 h in ANXA1-overexpressing PK15 and IPEC-J2 cells. (C) Schematic design of CRISPR-resistant ANXA1 (pANXA1). (D) Western blot confirming ANXA1 restoration in PK15-ANXA1KO after PAstV infection. (E) RT-qPCR analysis of PAstV infection (MOI = 0.01) at 24 h in ANXA1-rescued PK15 cells. (F) RT-qPCR analysis of PAstV infection (MOI = 0.01) at 24 h in ANXA1 polyclonal KO IPEC-J2 cells. (G) Immunofluorescence detection of virus replication in PK15-WT and PK15-ANXA1 KO cells. (H) RT-qPCR analysis of virus replication (MOI = 0.1) in PK15-ANXA1 KO cells at 24 h post-infection. (I, J) Western blot (I) and RT-qPCR (J) analysis of ANXA1 expression in PK15 cells infected with PAstV (MOI = 1). Data represent mean ± SD (n = 3). Statistical significance by unpaired two-tailed Student’s t-test and Two-way ANOVA. (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).
Fig 4.
ANXA1 binds directly to the PAstV Capsid acidic domain via its repeat III domain.
(A) Co-immunoprecipitation assay of ANXA1-HA and Capsid -Flag co-transfected in 293T cells, followed by Western blot detection (n = 2). (B) Confocal microscopy showing colocalization of ANXA1-HA and Capsid-Flag. Scale bar, 5 μm. (C) Predicted interaction interface between ANXA1 and Capsid by protein-protein docking using HDOCKlite v1.1. (D) PLA signal (orange) showing interaction between ANXA1 and Capsid in PK15 cells infected with PAstV (MOI = 1) for 24 h. Scale bar, 5 μm. (E) Schematic of ANXA1 conserved C-terminal domain composed of four annexin repeats, recombinant ANXA1 plasmids with HA-tag. (F) Flow cytometry analysis of PAstV-ILOV infection (MOI = 1) in PK15-ANXA1 KO cells transfected with different ANXA1 recombinant plasmids. Data represent mean ± SD (n = 3). Statistical analysis by unpaired two-tailed Student’s t-test (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001). (G) Schematic depicting PAstV VP90 maturation process. (H) Molecular dynamics simulation of ANXA1-ΔR3 and Capsid acidic domain. (I, J) SPR analysis of ANXA1-ΔR3 binding to Capsid-acidic domain.
Fig 5.
ANXA1 modulates apoptosis, orchestrates RIG-I-mediated antiviral signaling, and pharmacological targeting of ANXA1 impairs PAstV infection ex vivo.
WT and ANXA1-KO PK15 cells infected with PAstV (MOI = 0.01). At 12 hpi, cell lysates were analyzed by Western blot for cleaved caspase-3 (A) or by Annexin V/PI staining (B) for apoptosis. (C) Venn diagram showing differentially expressed genes between PK15-WT and PK15-ANXA1 KO cells. (D) KEGG pathway analysis of differentially expressed genes. (E) Western blot of indicated proteins at specified time points (n = 2). (F, G) RT-qPCR analysis of PAstV infection (MOI = 0.01, 24 h) in PK15 and IPEC-J2 cells after ODN transfection (F) or WRW4 treatment (G). Data represent mean ± SD (n = 3). Statistical analysis by unpaired two-tailed Student’s t-test (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).
Fig 6.
A working model for the dual-stage roles of ANXA1 during PAstV infection.
Upper panel: At the cell surface, ANXA1 promotes PAstV attachment. The PAstV virion is depicted as a non-enveloped capsid. Inset, the R3 repeat of ANXA1 engages the acidic domain within the ORF2 capsid. Additional, as-yet-unidentified attachment factors or receptors may contribute to PAstV binding. Lower panel: Following infection, PAstV genomic RNA is sensed in the cytosol by RIG-I, triggering MAVS-dependent signalling and downstream activation of IRF3, culminating in induction of IFN-β transcription and type I interferon output. ANXA1 enhances this RIG-I–IRF3 axis. Dashed lines indicate proposed or indirect interactions supported by the data, and arrows indicate the direction of events. This figure was created with FigDraw (license code: YPWIR12153).